The study of human cells for medical screening, diagnostic and other medical purposes is well known. For example, red blood cell count, mean cell volume (MCV), hemoglobin content and hematocrit are well known and commonly employed red blood cell parameters used in medical study and patient care.
The red blood cell is an excellent osmometer in that the cell will change in shape and volume, depending on the osmotic pressure or chemical nature of the fluid surrounding it. If the red blood cell is suspended in a solution of the same osmotic pressure as that of the intracellular fluid, the suspending fluid is said to be isotonic. Should the red blood cell be suspended in a hypotonic suspending solution, the red blood cell will swell and may even rupture due to the water taken in, in an effort to balance the internal osmotic pressure to that of the suspending solution. Other cells of the blood, namely the white blood cells and platelets, will act in much the same manner.
It is also well known that the osmotic pressure of solutions, i.e., their osmolality, such as saline solutions, varies with their concentration and types of solutes, in that the difference between the osmotic pressure within a cell and that of its suspension liquid causes the previously described change in volume and also a change in electrical resistance. In addition to a hypotonic solution, other volume changing solutions are known in the art. For instance, when red blood cells are exposed to hemolytic agents such as saponin, their membrane lipids are altered, so that water is allowed to enter the cell, causing the cell to swell and eventually rupture. If an excess of lytic agent is used, the red cell may be completely ruptured into extremely small fragments.
The above-described facts have been used extensively in the measurement of the degree of fragility of the red blood cells to changes in osmotic pressure. In several clinical disorders where the erythropoietic system is involved red blood cells demonstrate an increased or decreased fragility, depending on the specific disorder. An example of such a disorder is Thalassemia, where the cell fragility is decreased.
One apparatus of the prior art used for measuring osmotic fragility is the Fragiligraph.TM. sold by Manufacturer Reference. Using this apparatus, classic osmotic fragility curves have been generated by forming an increasingly hypotonic solution. A portion of the blood sample is introduced into each solution, and after a fixed time period, the degree of hemolysis is measured optically for each solution. Hemolysis is a process which is characterized by the release of the hemoglobin from the red blood cells, either by the pores of the membrane opening up or by ruptures in the membrane. From the optical measurements for each of the solutions, the percentage of hemolysis is plotted against the decreasing concentration of sodium chloride. Variations in the generated curve shapes have been recognized and related to different health conditions.
Cell and particle counting and measuring instruments, examples being those sold under the trademark Coulter Counter.RTM. by Coulter Electronics, Inc., Hialeah, Fla., employ electronic sensing means which directly respond to the electrical resistance of each cell to count and measure each cell and progressively record cell parameters of a sample of cells in an isotonic solution. The Coulter Counter.RTM. particle measuring instruments operate upon the well-known and documented principle of particle and cell measurement employing a sensing aperture path, which also is disclosed in Coulter U.S. Pat. No. 2,656,508 and improvement U.S. Pat. No. 3,259,842. A form of MCV measuring apparatus especially useful with a Coulter Counter.RTM. instrument is taught in U.S. Pat. No. 3,473,010. The response of a Coulter Counter.RTM. electric sensor is influenced at least by the shape, deformability and flow rate of the microscopic item being measured as it flows through the sensing aperture path. Since cells are subject to some deformation as they pass through the sensing aperture path, their electrical resistance measurement and their measured volume may differ from their true volume. To distinguish between true volume and measured volume, the term "apparent volume" will be employed herein to refer to measured volume. It is also well-known that as the cells swell and their pores expand, the cell will be more conductive of the current so that its apparent volume will decrease with respect to its true volume.
The above-described Coulter Counter.RTM. has been used to study white blood cells in a hypertonic solution, as shown in the article entitled "The Permeability of the Lymphocyte Membrane: Applying a Particle Size Analyzer and a Hybrid Computer to Measure Rapid Changes in Cell Volume", by Harold G. Hempling, Acta Cytologica, Vol. 21, No. 1, 1977. A hypertonic solution has the opposite effect on the cells, in that the increase in sodium chloride over that found in a normal isotonic solution causes the cell to shrink and its volume and electrical resistance to decrease.